Delivery of chromosomes, the basic units of inheritance, to each daughter cell during cell division is mediated by the centromere. Unlike typical genes, in metazoans this central genetic element is not determined by DNA sequence. Rather, functional centromeres are determined epigenetically through stable acquisition of an unexplained, non-DNA mark. A prime candidate for a component of such an epigenetic mark is CENP-A, a histone H3 variant found exclusively at functional centromeres and which we have previously demonstrated to assemble into conformationally more constained, functionally divergent nucelsomes. We will now determine if the structural rigidity and compactness of nucleosomes targeted to the centromere is sufficient to maintain centromere identity and function in mammalian cells. Using an in vivo fluorescence pulse-chase tagging method we identified, we will determine the timing of loading of constitutive components required for CENP-A loading, as well as the DNA targets for centromre components CENP-T and CENP-W. To identify how centromeric chromatin is replicated, we will identify cell cycle-dependent covalent modification to CENP-A and its assembly chaperone HJURP, and will identify how timing and loading is altered in cells with reduced HJURP. Following our discovery that CENP-A and other components known for essential roles in centromere assembly are rapidly recruited to sites of DNA damage, we will assess the roles of CENP-A and its partners in DNA repair. This will include determination of the mechanism of transient CENP-A recruitment to sites of DNA damage and the extent of chromatin remodeling following DNA damage. Finally, methods will be developed for rapidly following de novo centromere assembly in mammalian cells after introduction of large arrays of centromeric alphoid DNA carried on bacterial or yeast artificial chromosomes. These will be used to identify factors critical for formation of new centromeres.